US5612196A - Human serun albumin, preparation and use - Google Patents

Human serun albumin, preparation and use Download PDF

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US5612196A
US5612196A US08/256,966 US25696694A US5612196A US 5612196 A US5612196 A US 5612196A US 25696694 A US25696694 A US 25696694A US 5612196 A US5612196 A US 5612196A
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plasmid
hsa
gene
sequence
promoter
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J er ome Becquart
Reinhard Fleer
G erard Jung
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Novozymes Biopharma DK AS
Albumedix Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a preparation of human serum albumin, a process for producing it, and its uses.
  • HSA Human serum albumin
  • the genes encoding HSA are known to be highly polymorphic, and more than 30 apparently different genetic variants have been identified by electrophoretic analysis under various conditions (Weitkamp, L. R. et al., Ann. Hum. Genet. 37 (1973) 291-226).
  • the gene for HSA is cut in 15 exons by 14 intron sequences and comprises 16,961 nucleotides, from the supposed "capping" site up to the first site for addition of poly(A).
  • Human albumin is synthesized in the liver hepatocytes and then secreted in the blood stream. This synthesis leads, in a first instance, to a precursor, prepro-HSA, which contains a signal sequence of 18 amino acids directing the nascent polypeptide in the secretory pathway.
  • HSA is the most abundant protein in the blood, with a concentration of about 40 g per liter of serum. There are therefore about 160 g of albumin circulating in the human body at any time.
  • the most important role of HSA is to maintain a normal osmolarity of the blood stream. It also has exceptional binding capacity for various substances and plays a role both in the endogenous transport of hydrophobic molecules (such as steroids and bile salts) and in that of various therapeutic substances which can also be transported to their respective sites of action.
  • hydrophobic molecules such as steroids and bile salts
  • HSA has recently been implicated in the catabolism of prostaglandins.
  • HSA represents 40% of the world market for plasma proteins. Its commercial interest lies in the fact that this product is widely used, for example in so called replacement solutions to compensate for blood losses during surgical procedures, accidents or haemorrhages, and at doses which may be as high as several tens of grams per day per individual. Currently, the annual consumption of HSA can be estimated at more than 300 tonnes.
  • the HSA available on the market is produced by purification from biological material of human origin.
  • it is obtained by conventional techniques for fractionation of plasma obtained from blood donations (Cohn et al., J. Am. Chem. Soc. 68 (1946) 459 pp), or by extraction from human placenta, according to the technique described by J. Liautaud et al. (13th International Congress of IABS, Budapest; A: "Purification of proteins. Development of biological standard", Karger (ed), Bale, 27 (1973) 107 pp).
  • the first genetic engineering experiments used the bacterium E. coli as host organism.
  • European Patents EP 236 210, EP 200 590, EP 198 745 or EP 1 929 describe processes for the production of HSA in E. coli using various expression vectors, various transcriptional promoters, and various signals for secretion. Subsequently, work relating to the secretion of HSA in Bacillus subtilis was also carried out, even though the levels of albumin obtained in this system still do not appear to be satisfactory (Saunders et al., J. Bacteriol. 1.69 (1987) 2917).
  • the recombinant HSA should possess the physico-chemical properties of native albumin and meet certain criteria of homogeneity, purity and stability.
  • the pharmacopoeia sets a certain number of parameters for solutions of plasma albumAn, namely a pH value, a protein content, a content of polymers and aggregates, a content of alkaline phosphatase, of potassium and of sodium and a certain protein composition. It also requires a certain absorbance, the conformity with a test of sterility, with an assay for pyrogens and for toxicity (see "Albumini humani solutio" European pharmacopoeia (1984) 255).
  • Example B1 illustrates the problem of coloration in the case of an expression system using, under the conditions of the prior art, a yeast as host organism.
  • Example B1 is not restrictive but illustrates a coloration phenomenon which was observed for all the strains tested, regardless of the mode of fermentation adopted (fed-batch, batch, continuous). Moreover, in spite of the numerous efforts made to this end, it has never been possible to remove this coloration by purification (see Example B2).
  • One of the aspects of the invention is to provide a preparation of recombinant HSA of good quality, possessing the properties of extracted HSA, and which can be used in the pharmaceutical field.
  • one aspect of the invention is to provide a preparation of recombinant HSA possessing, after purification by known methods (concentration, precipitation, chromatography and the like), a colorimetry index of less than 0.2.
  • One objective of the invention is to permit the production, in industrial quantities and at an economically profitable level, of a preparation of recombinant HSA which can be used pharmaceutically.
  • the applicant has now shown that it is possible to obtain a non-coloured HSA solution in industrial quantities by the recombinant route.
  • the present invention rests on the demonstration that the quality of the HSA produced is not only linked to the host or to the chosen vector, or to the process for the purification of HSA from the medium, but to a large extent to the very composition of the production medium.
  • the quality of the HSA produced is not only linked to the host or to the chosen vector, or to the process for the purification of HSA from the medium, but to a large extent to the very composition of the production medium.
  • a first subject of the invention therefore lies in a human serum albumin characterized in that it possesses a colorimetry index of less than 0.2 and in that it results from the expression, in a eucaryotic or procaryotic host, of an exogenous DNA sequence.
  • the present invention provides, for the first time, a recombinant HSA having a colorimetry index of less than 0.2. It thus provides a recombinant HSA which can be used in the pharmaceutical field, with very low risks of immunogenic reactions. Furthermore, compared with plasma HSA, the HSA of the invention offers the advantage of being homogeneous and of perfectly defined composition. Indeed, because of its polymorphism, numerous HSA variants exist. Thus, among those which have been identified, some variants conserve a substituted pro peptide (Brennen and Carrell, Nature 274 (1978) 908; Galliano et al., Rev. Fr. Transfus. Immuno-H ematol.
  • the plasma HSA obtained by extraction of biological material derived from a very large number of human donors potentially contains all the HSA variants resulting from its polymorphism.
  • the present invention provides a homogeneous and defined HSA, because of its preparation by the genetic route, by expression of one or more identified DNA sequences.
  • the HSA of the present invention has a colorimetry index of less than 0.15.
  • the HSA of the present invention has a colorimetry index of less than 0.1.
  • Example B5 Other physico-chemical characteristics of the HSA of the present invention are given in Example B5, namely especially its fluorescence spectrum. All these parameters demonstrate the quality of the HSA of the invention.
  • exogenous DNA sequence is understood to mean any DNA sequence introduced artificially into the host used and encoding HSA. In particular, it may be, depending on the host, genomic sequences, cDNA, hybrid sequences and the like. For a better implementation of the invention, the use of a cDNA is however preferred. Such sequences have already been described in the prior art (cf especially EP 361 991 and Dugaiczyk et al., J. Supramol. Struct. & Cell Biochem., Suppl. 5 (1981)). Moreover, this exogenous DNA generally comprises a region for initiation of transcription and translation joined to the 5 terminal end of the coding sequence, so as to direct and regulate the transcription and translation of the said sequence. The choice of these promoter regions may vary according to the host used.
  • the exogenous DNA is preferably part of a vector, which may be one affording autonomous or integrarive replication.
  • autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host.
  • this may be replication origins derived from plasmids: pKD1 (EP 241 435), 2 ⁇ (Beggs, Nature 275 (1978) 104-109), and the like; or alternatively chromosomal sequences (ARS).
  • integrarive vectors these can be prepared for example using sequences homologous to certain regions of the host genome, which permit, by homologous recombination, the integration of the vector.
  • the use of rDNA permits a multiple integration of the exogenous DNA, and therefore its presence in higher copy number per cell.
  • the HSA of the invention results from the expression, in an eucaryotic or procaryotic host, of an exogenous DNA sequence and from the secretion of the expression product of the said sequence into the culture medium. It is indeed particularly advantageous to be able to obtain, by the recombinant route, a HSA of pharmaceutical quality directly in the culture medium.
  • the exogenous DNA sequence comprises, upstream of the sequence encoding HSA, or, where appropriate, between the region for initiation of transcription and translation and the coding sequence, a "leader" sequence directing the nascent protein in the secretory pathways of the host used.
  • This "leader” sequence may be the natural "leader” sequence of HSA, but it may also be a heterologous sequence (derived from a gene encoding another protein) or even artificial. The choice of one of these sequences is especially guided by the host used. As example, when the host used is a yeast, it is possible to use, as heterologous "leader” sequence, that of the pheromone factor ⁇ , invertase or acid phosphatase.
  • yeasts there may be mentioned animal cells, yeasts or fungi.
  • yeasts there may be mentioned yeasts of the genus Saccharomyces, Kluyveromyces, Pichia pastoris, Schwanniomyces or Hansenula.
  • animal cells there may be mentioned COS, CHO and C127 cells and the like.
  • fungi capable of being used in the present invention there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
  • Another subject of the invention relates to a process for preparing HSA having a colorimetry index of less than 0.2 according to which the following steps are carried out:
  • an exogenous DNA encoding the human serum albumin under the control of transcriptional and translational signals appropriate to the host used is introduced into a eucaryotic or procaryotic host cell,
  • the cell thus obtained is cultured in a medium of defined composition containing at least one carbon source chosen from alcohols, non-reducing sugars, organic acids or glucose derivatives substituted on the oxygen of the carbon C4; or in a medium prepared so as to remove or limit the formation of aldehyde type impurities, and,
  • the HSA produced is recovered.
  • the exogenous DNA can be introduced into the host cell by various techniques.
  • the introduction can be carried out by transformation, conjugation or electroporation.
  • transformation various procedures have been described in the prior art. In particular, it can be carried out by treating the whole cells in the presence of lithium acetate and polyethylene glycol according to the technique described by Ito et al. (J. Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and dimethyl sulphoxide according to the technique of Durrens et al. (Curr. Genet. 18 (1990) 7).
  • An alternative procedure has also been described in Patent Application EP 361 991. More specifically, for the procaryotic cells, the transformation can be carried out according to the technique described by Dagert et al. (Gene 6 (1979) 23-28) by treating with a solution of CaCl 2 followed by heat shock. For animal cells, it can also be carried out by the calcium phosphate technique according to Haynes (Nucleic Acids Res., 11 (1983) 687-706).
  • eucaryotic hosts which can be used in the process of the invention, there may be mentioned the animal cells, yeasts or fungi which were mentioned above.
  • procaryotic hosts any bacterium defined above can be used.
  • the process is carried out using, as host cell, a eucaryotic cell.
  • the process of the invention is carried out using, as host cell, a yeast.
  • the exogenous DNA encoding the HSA which can be used in the process is defined as above.
  • it is a cDNA, a genomic DNA or a hybrid DNA.
  • this exogenous DNA generally comprises a region for initiation of transcription and translation joined to the 5' terminal end of the coding sequence, so as to direct and regulate the transcription and translation of the said sequence. The choice of this region may vary as a function of the host used.
  • the exogenous DNA comprises, upstream of the sequence encoding mature HSA, a "leader" sequence directing the nascent protein in the secretory pathways of the host used.
  • a "leader" sequence directing the nascent protein in the secretory pathways of the host used.
  • sequences have been defined above.
  • the exogenous DNA is preferably part of a vector, which may be one affording autonomous or integrated replication, as indicated above.
  • the process of production is characterized in that the HSA is secreted into the control medium. It is indeed particularly advantageous to be able to obtain, by the recombinant route, a HSA of pharmaceutical quality directly from the control medium.
  • the second step of the process of the invention consists in culturing the recombinant cells under conditions permitting the expression of the exogenous DNA sequence encoding HSA. This step is particularly important since it directly influences the quantity and quality of the HSA produced.
  • the present invention describes, for the first time, culture conditions which permit the production of a serum albumin of pharmaceutical qualities.
  • non-reducing sugars there may be mentioned for example sucrose, and, as organic acids, acetates or lactates.
  • the glucose derivatives which can be used in the present invention correspond more specifically to the following formula: ##STR1## in which R is different from hydrogen.
  • disaccharides and preferably the disaccharides having a glycoside bond of the 1-4 type such as maltose, cellobiose or lactose.
  • the process of the invention can also be implemented in a medium prepared so as to eliminate or limit the formation of aldehyde type impurities.
  • This type of preparation is generally useless when one or more of the carbon sources listed above are used in a medium of defined composition.
  • Various means can be used to limit the formation of such impurities, of which the choice depends on the medium (nature of the carbon source) and the host considered. As example, it may be advantageous to sterilize the carbon source at cold temperature (for example by filtration as illustrated in Example B4).
  • the third step of the invention makes it possible to extract, from the culture medium, the HSA produced.
  • this extraction can be carried out directly from the culture supernatent obtained by centrifugation.
  • This prior step can be carried out by various physical or physico-chemical means (sonication, grinding, osmotic shock, heat shock and the like).
  • the extraction can be carried out by various techniques known to a person skilled in the art and described in the literature. Generally, these techniques involve concentration, precipitation and chromatographic steps. Some of these various techniques are illustrated in the examples.
  • Another subject relates to any pharmaceutical composition comprising HSA as defined above.
  • FIG. 1 Construction of the hybrid promoter PGK/GAL.
  • FIG. 2 Strategy for the construction and representation of the vector pYG401.
  • FIG. 3 Construction and representation of the plasmid pP4-33.
  • FIG. 4 Construction and representation of the plasmid pYG65.
  • FIG. 5 Construction and representation of the plasmid pYG70.
  • FIG. 6 Construction and representation of the plasmid pYG72.
  • FIG. 7 Construction and representation of the plasmid pYG404 ⁇ HindIII.
  • FIG. 8 Construction and representation of the plasmid pYG128.
  • FIG. 9 Construction and representation of the plasmids pYG131 and pYG132.
  • FIG. 11 Strategy for the construction of the plasmid pYG70-2.
  • FIG. 12 Sequence of the synthetic oligodeoxynucleotides A-F having served for the construction of the adaptors 1 to 3.
  • FIG. 14 Strategy for the construction of the plasmid pYG1003. For the legend, see FIG. 11.
  • FIG. 15 Strategy for the construction of the plasmid pYG1023. For the legend, see FIG. 11.
  • FIGS. 16 and 17 UV spectra for various preparations of albumin: FIG. 16: (1) albumin BCQ759 of Examples B1 and B2; (2) albumin BCQ804LE of Example B5; (3) control albumin (IM). FIG. 17: albumin BCQ835GE of Example B5.
  • FIGS. 18 and 19 Fluorescence spectra for various preparations of albumin at 430 nm (FIG. 18) and 360 nm (FIG. 19).
  • Site-directed mutagenesis in vitro by oligodeoxynucleotides is carried out according to the method developed by Taylor et al. (Nucleic Acids Res. 13 (1985) 8749-8764) using the kit distributed by Amersham.
  • the sequencing of nucleotides is carried out according to the dideoxy technique described by Sanger et al. (Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5463-5467).
  • Enzymatic amplification of specific DNA fragments is carried out by the PCR reaction ("Polymerase-catalyzed Chain Reaction") under the conditions described by Mullis and Faloona (Meth. Enzym., 155 (1987) 335-350), and Saiki et al (Science 230 (1985) 1350-1354), using a "DNA thermal cycler" (Perkin Elmer Cetus) following the recommendations of the manufacturer.
  • a human serum albumin expression vector was prepared from the plasmid pYG19 (EP 361 991). The latter comprises the following elements:
  • the vector pYG401 was constructed from the plasmid pYG19 by modification at the level of the human serum albumin expression cassette.
  • the albumin gene is no longer under the control of the promoter of the PGK gene of S. cerevisiae, but under the control of a hybrid promoter between the promoters of the PGK and GAL1/GAL10 genes of S. cerevisiae.
  • This hybrid promoter was obtained by replacing the UAS ("Upstream Activating Sequence") region of the PGK promoter (Stanway et al., Nucl. Acid Res. 15 (1987) 6855) with the UAS region of the GAL1/GAL10 promoter (johnston and Davies, Mol. Cell. Biol. 4 (1984) 1440; West et al., Mol. Cell. Biol. 4 (1984) 2467).
  • This hybrid promoter was constructed in the following manner (cf FIG. 1):
  • This plasmid contains the promoter of the PGK gene S. cerevisiae isolated from the plasmid pYG19 in the form of a SalI-HindIII fragment, and cloned into the bacteriophage M13mp18. It was then modified by site-directed mutagenesis in order to introduce the following restriction sites:
  • the UAS of the GAL1/GAL10 promoter was isolated from the plasmid pG1 described by Miyajima et al. (Nucl. Acid. Res 12 (1984) 6397-6414; Cloning Vectors, Pouwels et al., Elsevier (1985) VI-B-ii-2). This plasmid is deposited at ATCC under the number 37305.
  • the plasmid pG1 contains a fragment of 0.8 kb containing the GAL1/GAL10 promoter of S. cerevisiae, inserted into the HindII site of the plasmid pUC8, from which it can be excised in the form of a BamHI-PstI fragment (FIG. 1).
  • This fragment was excised from pG1, purified and then digested with the enzymes RsaI and AluI, whose respective cleavage sites are localized on either side of the UAS region.
  • a 143 bp fragment was thus isolated by electroelution and then converted to a NotI fragment by adding a linker 5'-GCGGCCGC-3'(SEQ ID NO:7). This fragment was then cloned into the plasmid pYG29, previously digested with NotI.
  • pYG32 (FIG. 1).
  • the SalI-HindIII fragment carrying the hybrid promoter was isolated from pYG32 and ligated to a synthetic HindIII-BstEII adaptor composed of the following 2 complementary strands: 5'-AGC TTT ACA ACA AAT ATA AAA ACA ATG AAG TGG-3' (SEQ ID NO:8) and 5'-GT TAC CCA CTT CAT TGT TTT TAT ATT TGT TGT AA-3' (SEQ ID NO:9) (the transcriptional initiation codon is represented in bold characters).
  • This adaptor reconstitutes the 22 bp situated immediately upstream of the PGK structural gene of S. cerevisiae, and comprises the first codons of the gene encoding preproHSA, up to a BstEII site present in the native gene (FIG. 1).
  • the human albumin expression cassette was then reconstituted by ligating the SalI-BstEII fragment thus obtained carrying the hybrid promoter and the 5' end of the albumin structural gene, to the BstEII-SacI fragment isolated from the plasmid pYG19, carrying the rest of the albumin gene, and the terminator of the PGK gene of S. cerevisiae (FIG. 2).
  • the cassette thus obtained was used to replace the SalI-SacI expression cassette carried by the plasmid pYG19.
  • the resulting vector is called pYG401 (FIG. 2).
  • the promoter K1ADH4 was obtained by screening a total genomic DNA library for Kluyveromyvces lactis CBS2359/152 by means of a heterologous probe obtained from the ADH2 gene of S. cerevisiae. More specifically, the library was obtained by cloning into the BamHI site of the replacement vector Lambda-L47 the product of a partial digestion with the enzyme Sau3A of DNA of K. lactis CBS2359/152.
  • the probe used for the hybridization is a 980 bp EcoRV-BamHI fragment comprising the region encoding the ADH2 structural gene of S. cerevisiae, with the exception of the first 70 bp (probe A).
  • This fragment is obtained by enzymatic digestion from a plasmid called pBR322.ADR2.BSa (Williamson et al., Cell 23 (1981) 605-614; Russell et al., J. Biol. Chem. 258 (1983) 2674-2682).
  • a fragment of about 8 kb was thus isolated and subcloned into the BamHI site of the plasmid pBR322 in order to generate the plasmid p6-4-98 (FIG. 3).
  • the BamHI insert carried by this plasmid was then mapped by means of restriction enzymes, and the promoter region of the K1ADH4 gene was localized on this fragment by differential hybridizations using the probe A as well as a second probe corresponding to the BamHI-EcoRV fragment of about 1100 bp of the plasmid pBR322.ADR2.BSa (probe B).
  • the plasmid p6-4-98 was digested with the enzyme HindIII and a 2.2 kb fragment was isolated. This fragment was then purified by standard techniques and subcloned into the HindIII site of the plasmid pTZ19 in order to generate the plasmid p6-2200-2 (FIG. 3). Analysis of the subcloned fragment reveals that it contains the first 14 codons of the K1ADH4 gene and the region situated upstream thereof, comprising the elements for regulating the expression.
  • the part between the BglII site and the translational initiation codon ATG was sequenced using the chain termination method (Sanger et al., Proc. Nat. Acad. Sci. 74 (1977) 5463).
  • a portable promoter was prepared by inserting into the 2.2 kb HindIII fragment present in the plasmid p6-2200-2 a BamHI restriction site at position -16 relative to the ATG codon of the K1ADH4 gene.
  • the BamHI site was introduced at position -16 relative to the site for initiation of translation (ATG) of the K1ADH4 gene by site-directed mutagenesis using the double primer technique (Sambrook, Fritsch, Maniatis, Molecular Cloning Laboratory Manual, Cold Spring Harbor Lab Press, 1989).
  • the sequence of the synthetic oligodeoxynucleotides used for this mutagenesis is given below.
  • pP4-33 The plasmid thus obtained is called pP4-33 (FIG. 3).
  • HSA human serum albumin
  • HSA prepro-human albumin
  • a replicon and a selectable marker (bla gene conferring the resistance to ampicillin) for E. coli.
  • This plasmid differs from pYG404 only in the destruction of the HindIII site, localized in the aph gene, by site-directed mutagenesis. This modification then made it possible to substitute the LAC4 promoter present in the plasmid pYG404 ⁇ H in the form of a SalI-HindIII fragment by the promoter K1ADH4 also constructed in the form of a SalI-HindIII fragment.
  • pYG72 In order to carry out the deletion of the HindIII site in the cloning vector pYG404, various subcloning steps were carried out, giving rise to an intermediate construct: pYG72 (FIG. 6).
  • This vector corresponds to the plasmid pKan707 (EP 361 991) in which the SacI fragment containing the URA3 gene has been removed as well as the unique HindIII site present in the aph gene.
  • the 1.3 kb PstI fragment carrying the aph gene was subcloned from the plasmid pKan707 into the bacteriophage M13mp7 in order to give the vector pYG64 (FIG. 4).
  • the HindIII site was destroyed by site-directed mutagenesis (Cf general cloning techniques) using the following oligodeoxynucleotide: 5'-GAAATG CAT AAG CTC TTG CCA TTC TCA CCG-3' (SEQ ID NO:13), permitting the replacement of the CTT triplet encoding leucine 185 with the triplet CTC. This change does not modify the resulting protein sequence.
  • the plasmid obtained was called pYG65 (FIG. 4).
  • the part containing the bacterial replicon of the vector pKan707 was isolated by digestion with the enzyme EcoRI and recircularization with T4 DNA ligase, generating the intermediate plasmid pYG69.
  • the PstI fragment present in the latter containing the aph gene was then replaced by the equivalent mutated fragment obtained from the plasmid pYG65.
  • This construct was called pYG70 (FIG. 5).
  • the 4.7 kb pKD1 sequence between the EcoRI and SacI sites was then introduced into this vector in order to obtain pYG72 (FIG. 6).
  • the vector pYG404 ⁇ H was obtained by inserting the expression cassette obtained from the plasmid pYG404 (EP 361 991) in the form of a SalI-SacI fragment at the corresponding sites of pYG72 (FIG. 7).
  • the promoter K1ADH4 carried on the BglII-BamHI fragment obtained from the plasmid pP4-33 (Cf 2.1.) was modified in the following manner in order to adapt it for use in expression vectors derived from the plasmid pYG404 ⁇ H:
  • the 1.2 kb fragment carrying the promoter K1ADH4 was isolated from an agarose gel and subcloned into the vector pBC SK+ (Stratagene, La Jolla, Calif., U.S.A.) previously linearized with the enzyme ClaI and treated with ⁇ Mung Bean ⁇ nuclease as well as with calf alkaline phosphatase (CIP).
  • the plasmid obtained in this manner (pYG128, FIG. 8) permits the isolation of the promoter K1ADH4 in the form of a 1.2 kb SalI-HindIII fragment.
  • the 8.7 kb SalI-HindIII fragment containing the pKD1 part and the selectable markers as well as the 1.9 kb HindIII-HindIII fragment carrying the gene encoding prepro-HSA were isolated from the vector pYG404 ⁇ H and religated in the presence of the 1.2 kb SalI-HindIII fragment obtained from the plasmid pYG128 and carrying the promoter K1ADH4. Two plasmids were obtained in this manner:
  • pYG131 (FIG. 9), corresponding to a cloning vector permitting the insertion, at the unique HindIII site, of any gene which it is desired to express under the control of the promoter K1ADH4, and
  • pYG132 (FIG. 9), which is identical to the plasmid pYG131 except that it contains the preproHSA gene introduced into the HindIII site.
  • the PGK gene was isolated from K. lactis CBS2359 by the screening of a partial genomic library with a heterologous probe corresponding to the N-terminal part of the PGK gene of S. cerevisiae (Dobson et al., Nucl. Acid. Res. 10 (1982) 2625-2637). More specifically, the probe used corresponds to the PvuI-EcoRI fragment of 1.3 kb.
  • the plasmid pYG70 described in FIG. 5 was modified as follows.
  • the plasmid pYG70 was digested with SphI, and the cohesive ends were then removed by digestion in the presence of phage T4 DNA polymerase. After ligation in the presence of ligase, the plasmid pYG70 ⁇ SphI was obtained (see FIG. 11).
  • the adaptor 1 was obtained by hybridization of the synthetic oligodeoxynucleotides A and B represented in FIG. 12. For that, 2 ⁇ g of each oligodeoxynucleotide were incubated in a hybridization buffer qs 20 ⁇ l (30 mM Tris-HCl buffer pH 7.5; 30 mM NaCl; 7.5 mM MgCl 2 ; 0.25 mM ATP; 2 mM DTT; 0.2 mM EDTA), and then the temperature was raised to 80° C. for 10 minutes, and brought back slowly to room temperature.
  • a hybridization buffer qs 20 ⁇ l (30 mM Tris-HCl buffer pH 7.5; 30 mM NaCl; 7.5 mM MgCl 2 ; 0.25 mM ATP; 2 mM DTT; 0.2 mM EDTA
  • the adaptor thus obtained contains the cleavage sites for the following enzymes: SacI; SalI; MluI; BssHII and SfiI, and makes it possible to remove, during its introduction, the SalI site present in the plasmid pYG70 ⁇ SphI.
  • This adaptor was introduced by ligation into the plasmid pYG70 ⁇ SphI previously digested with the enzymes SalI and SacI.
  • the plasmid obtained is called pYG70-1.
  • the adaptor 2 was prepared following the procedure described for the adaptor 1, using the oligodeoxynucleotides C and D described in FIG. 12.
  • This adaptor contains cleavage sites for the following enzymest SfiI; AatII; Sph; NarI and SacI and makes it possible to remove, during its introduction, the EcoRI site present in the plasmid pYG70-1. It was introduced, by ligation, into the plasmid pYG70-1 previously digested with the enzymes EcoRI and SacI, to form the plasmid pYG70-2 (FIG. 11).
  • the human serum albumin expression cassette used comprises:
  • This cassette was isolated from the plasmid pYG404 (EP 361 991) in the form of a SalI-SacI fragment, and then introduced by ligation into the plasmid pYG70-2 previously digested with the corresponding enzymes.
  • the plasmid obtained is called pYG70-3 (FIG. 13).
  • the K. lactis PGK gene was isolated from the plasmid pYG600 (FIG. 10), subcloned into the plasmid pYG1002 in order to generate the plasmid pYG1003, and then isolated from the latter in the form of an MluI-BssHII fragment.
  • the plasmid pYG1003 was obtained in the following manner (FIG. 14):
  • the plasmid pIC20H (Marsh et al., Gene 32 (1984) 481) was digested with BglII and EcoRI so as to introduce the adaptor 3.
  • This adaptor which provides the EcoRI, BssHII, ClaI, NheI, MluI and BglII sites, was obtained as described above (2.(a) (ii)), by hybridization of the oligodeoxynucleotides E and F (FIG. 12).
  • the resulting plasmid is called pYG1002.
  • the PGK gene of K. lactis was introduced into this new plasmid in the form of a ClaI-NheI fragment, derived from the plasmid pYG600.
  • the plasmid obtained is called pYG1003 (FIG. 14).
  • the MluI-BssHII fragment derived from the plasmid pYG1003 carrying the PGK gene of K. lactis was then introduced into the corresponding sites in the plasmid pYG70-3, to generate the plasmid pYG70-4 (FIG. 15).
  • the PGK gene of K. lactis is henceforth placed under the control of the bidirectional kl promoter of the Killer toxin.
  • the plasmids pYG70-4 (FIG. 15) and pKD1 (EP 361 991) were digested with SphI and ligated together in the presence of ligase. At the end of this ligation, 4 vectors can be obtained, depending on the conformation of the plasmid pKD1 (form A or form B) and the orientation of the part corresponding to the plasmid pYG70-4 relative to pKD1.
  • This vector comprises:
  • pKD1 an entire sequence of the plasmid pKD1, which makes pYG1023 a multicopy plasmid which is stable and capable of replicating in yeasts and preferably yeasts of the genus Kluyveromyces,
  • lactis pgk strain the mutated aph gene under the control of the bidirectional k1 promoter of the Killer toxin and the PGK gene of K. lactis under the control of the same promoter but transcribed divergently compared with the aph gene.
  • the culture was carried out in a 2 liter fermenter at 28° C. in fed-batch mode. Initially, the fermenter contains 0.7 liter of basic medium (glucose 2 g/l, yeast extract 3 g/l, salts). A 50 ml culture in exponential growth phase (inoculated from a frozen suspension) is used to inoculate the fermenter. After a preliminary batch-type growth phase, the additional load (glucose, corn steep, ammonium salts: 40%/13%/7% [w/v]) is added exponentially. After 64 hours of culture, the broth is centrifuged and the supernatent microfiltered on a 2 ⁇ membrane.
  • basic medium glucose 2 g/l, yeast extract 3 g/l, salts
  • a 50 ml culture in exponential growth phase inoculated from a frozen suspension
  • the additional load (glucose, corn steep, ammonium salts: 40%/13%/7% [w/v]) is added exponentially. After 64 hours of culture, the broth is centrif
  • the purification procedure comprises an affinity chromatrography step on Blue Trisacryl (IBF, France) from which the albumin is eluted by a high salt concentration (3.5M NaCl), and then, after concentration and diafiltration, a passage on a Q-sepharose "fast flow" type ion exchanger.
  • the HSA thus purified has a homogeneous band in SDS-PAGE electrophoresis and is indistinguishable from natural albumins taken as reference in numerous tests for biochemical characterization.
  • BCQ 759 50 mg are denatured by incubation in 2 ml of 7M guanidine-HCl and 0.3M ⁇ -mercaptoethanol. This solution is heated for 1 hour at 100° C. After cooling, 500 ⁇ l of denatured rHSA are injected into a TSK 3000SW column equilibrated with 8M urea and 0.3M ⁇ -mercaptoethanol in order to remove the small molecules which may be strongly attached (but not covalently) to HSA.
  • the tested media consist of a basic medium Bo defined as follows:
  • the three erlen flasks are incubated with HSA at 50 mg/l and stirred at 28° C. for 3 days.
  • the medium is centrifuged and filtered on a 0.2 ⁇ membrane. 5 ml of affinity support Blue Trisacryl M (IBF France) are added and the mixture is stirred gently. After 1 hour, the mother liquors are separated on sintered glass and the albumin eluted with a 3.5M NaCl solution. The eluate is then concentrated and diafiltered with water on a 10 kD ultrafiltration membrane.
  • affinity support Blue Trisacryl M IBF France
  • the results obtained after UV analysis are the following:

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WO1993015204A1 (fr) 1993-08-05
JPH07503137A (ja) 1995-04-06
AU3503893A (en) 1993-09-01
CA2126356A1 (fr) 1993-08-05
HUT70570A (en) 1995-10-30
NO942780L (no) 1994-07-26
NZ249153A (en) 1996-03-26
AU686996B2 (en) 1998-02-19
HU218353B (hu) 2000-08-28
EP0625202A1 (fr) 1994-11-23
FI943512A (fi) 1994-07-26
FR2686620B1 (fr) 1995-06-23
NO942780D0 (no) 1994-07-26
FI943512A0 (fi) 1994-07-26
DE69325794T2 (de) 2000-01-13
ATE182623T1 (de) 1999-08-15
DE69325794D1 (de) 1999-09-02
FR2686620A1 (fr) 1993-07-30
ES2137251T3 (es) 1999-12-16
DK0625202T3 (da) 2000-02-28
JP3390990B2 (ja) 2003-03-31
CA2126356C (fr) 2003-03-18
GR3030873T3 (en) 1999-11-30
NO310418B1 (no) 2001-07-02
HU9401960D0 (en) 1994-09-28
EP0625202B1 (fr) 1999-07-28

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